Nerve growth factor (NGF) is the best known member of the neurotrophin family of growth factors (M. Bothwell. (1995) Annu Rev Neurosci., 18, 223-53). In its active form, NGF is a dimeric protein, composed by two identical polypeptide chains, that can bind two different cellular receptors trkA and p75 (C. Wiesmann, A. M. de Vos. (2001) Cell Mol Life Sci.; 58, 748-59; X. L. He, K. C. Garcia. (2004) Science. 7, 870-5.). Interestingly, the activation of these receptors produces divergent results. Indeed, activation of trkA provides protection against apoptotic cell death, whereas p75 mediates apoptosis in some neuronal cells. trkA is a transmembrane tyrosine kinase receptor that transduces NGF signals. As found for other receptors of the tyrosine kinase family, the dimeric NGF protein induces trkA dimerization, which leads to the auto-phosphorylation of the receptor and to the activation of essential cellular pathways.
NGF regulates neuronal survival, promote neurite outgrowth, and up-regulates certain neuronal functions such as mediation of pain and inflammation. NGF is also important for the regulation of neurons during development and in neuron regeneration after injury. In principle NGF could be used in the treatment of several pathological states. Target diseases include neuropathies of NGF-dependent neurons, therapy of acute nervous system injury such as ischemic stroke and spinal cord injury, and neuroectoderm-derived tumors. However, several limitations preclude the use of proteins as therapeutic agents (H. U. Saragovi, M. C. Zaccaro. (2002) Curr Pharm Des. 8, 2201-16). These include (a) the high level of proteolytic degradation of proteins, (b) difficulties associated with their administration limit, (c) their restricted penetration of the central nervous system, and (d) their availability in limited amounts. Therefore, it is desirable to search for new neurotrophic factor mimetics to overcome the drawbacks related to the use of NGF. Several different approaches have been developed in this research area. In particular, attempts to design different classes of compounds aimed at (a) activating, directly or indirectly, Trk receptors, (b) enhancing the actions of NTs on Trk receptors, or (c) influencing NTs expression and secretion have been carried out.
Particularly attractive is the search for small molecules, peptidic or non-peptidic, that bind to selective receptors and either mimic or antagonize neurotrophin activity. Different alternative strategies have been used to achieve this scope (H. U. Saragovi, M. C. Zaccaro. (2002) Curr Pharm Des. 8, 2201-16; S. M. Massa, Y. Xie, F. M. Longo. (2003) J Mol Neurosci., 20, 323-6). These include the screening of natural products and the rational design of novel compounds. Among the natural products, the fungal non-peptide metabolite L-783,281 is able to induce trkA auto-phosphorylation (N. Wilkie, P. B. Wingrove, J. G. Bilsland, L. Young, S. J. Harper, F. Hefti, S. Ellis, S. J. Pollack S J. (2001) J Neurochem. 78, 1135-45). Although the mechanism of action of this compound is not fully elucidated, it is believed that it interacts with the intracellular portion of trkA. The rational design of trkA ligands was carried out by considering regions of the sequences of anti-trkA antibodies and of NGF. Investigations on the trkA antibody, named 5C3, have shown that it promotes receptor internalization and PI(3)K signalling (L. LeSauteur, N. K. Cheung, R. Lisbona, H. U. Saragovi. (1996) Nat Biotechnol., 14, 1120-2). However, no effect on neuronal cells has been reported. A peptidomimetic approach that integrates information derived from 5C3 with the trkA-binding properties of peptides derived NGF sequence has leaded to the identification of a novel compound denoted as D30, that binds to trkA but does not compete with NGF. This molecule also protects embryonic DRG neurons from apoptosis.
Many efforts have been made to design NGF mimetics using data available on this neurotrophin. Multiple techniques have been used to deduce which regions of the NGF protein interact with NGF receptors (H. U. Saragovi, M. C. Zaccaro. (2002) Curr Pharm Des. 8, 2201-16; S. M. Massa, Y. Xie, F. M. Longo. (2003) J Mol Neurosci., 20, 323-6). Valuable information has been derived from structural investigations on complexes between neurothrophins and Trk receptors and from mutagenesis studies (C. Wiesmann, A. M. de Vos. (2001) Cell Mol Life Sci.; 58, 748-59). Regions corresponding to the loops 1 (residues 29-35), the loop 2 (residues 40-49), the loop 4 (residues 91-97), and the N-terminus (residues 1-25) have been identified as potential candidates. However, it has been found that linear peptides derived from NGF fragments do not show any biological activity. Data on cyclic peptides based on the loop 1 sequence are somewhat unclear. These molecules apparently interact with the p75 receptors rather than trkA and act as partial NGF agonists in promoting survival but not neurite outgrowth in dorsal root ganglion neurons. Compounds with NGF agonist activity, although rather limited, have been obtained by making cyclic derivatives of the loop 4 sequence. In order to mimic the biological action of NGF, homodimeric cyclic peptides based on loop 4 have also been developed (U.S. Pat. No. 5,958,875). These compounds show an enhanced agonist activity when compared to their monomeric counterparts which corresponds approximately to 20-40% of the NGF, when used in the 40-60 nM concentration range (H. U. Saragovi, M. C. Zaccaro. (2002) Curr Pharm Des. 8, 2201-16).
However, the use of these peptides is still limited by their relatively low efficacy when compared to NGF.